Agric. Biol. Chem., 48 (8), 2017-2023, 1984 2017

Properties of Dismutation Catalyzing Enzymeof Pseudomonas putida F61 Nobuo Kato, Hisataka Kobayashi, Masayuki Shimao and Chikahiro Sakazawa Department of Environmental Chemistry and Technology, Tottori University, Tottori 680, Japan Received January 9, 1984

Twoforms of formaldehyde distinguishable on disc-gel electrophoresis were isolated from the cell-free extract of Pseudomonasputida ¥61. The mobilities on SDS-gel electrophoresis and the NH2-terminal amino acids (arginine) of the two species were identical. The COOH- terminal amino acid sequence was found to be -Ser-Gly-Lys. The enzyme was inhibited by carbonyl, reducing and sulfhydryl reagents. The enzyme catalyzed the cross-dismutation reaction between formaldehyde and an aldehyde, such as propionaldehyde, acrolein, butyraldehyde, isobutyraldehyde and crotonaldehyde. The enzyme also catalyzed a coupled oxidoreduction between an alcohol and an aldehyde (RCH2OH+ R CHO^RCHO+R CH2OH) without addition of an electron acceptor. Aliphatic alcohols and aldehydes of C2 to C4 were utilized in this reaction.

In the preceding study,1} we found an en- (E; formaldehyde dismutase, X; unknown zyme, which catalyzed dismutation of form- prosthetic group, and XH2; its reduced form.) aldehyde to form equimolar amounts of meth- Furthermore, we have found that the enzyme anol and formic acid, in Pseudomonas putida catalyzes a unique alcohol: aldehyde oxido- F61. The enzyme, given the trivial name of reduction reaction: formaldehyde dismutase, was purified and RCH2OH+R CHO >RCHO+R CH2OH partially characterized. On the other hand, mammalian alcohol dehydrogenase (EC In this work, we describe more detailed 1.1.1.1) was reported to catalyze formalde- properties of this enzyme and its hyde dismutation on the addition of a cata- specificities in the cross-dismutation and al- lytic amount of NAD+or one of its deriva- cohol: aldehyde oxidoreduction reactions. tives.2) The enzyme from P. putida F61, which is distinct from the mammalian one, catalyz- MATERIALS AND METHODS ed the dismutation without addition of an electron acceptor, such as a pyridine nucleo- Organism and cultivation. P. putida F61 was grown on a tide coenzyme or artificial dye.X) Another uni- nutrient medium as reported previously.1* The harvested que property of the formaldehyde dismutase cells were washed twice with lOmMpotassium phosphate buffer, pH 7.0, and the cell paste was stored at -15°C was the catalytic activity of cross-dismutation until use. between two different aldehydes: RCHO"+H2O+Eà"X >RCOOH+Eà"XH2 Enzymeassay. Formaldehyde dismutase was assayed by determination of formic acid formed by means of pHstat R CHO+Eà"XH2 >R CH2OH+Eà"X titration with NaOHat pH 7.O.1* Protein determination. Protein was estimated by the Bio- RCHO+H2O+R CHO > Rad Protein Assay (Bio-Rad Laboratories) with bovine RCOOH+R CH2OH serum albumin as the standard. The protein concentra- tion of the purified enzyme was determined with an 2018 N. Kato et al.

4280nmvalue of 8.2 which was obtained from the absor- raise the yield of the purified enzyme, the purification bance and dry weight determinations. procedures were modified as follows. Amino acid determination. For determination of the (0 Acetone fractionation. The protamine-treated en- amino acid composition, the purified enzyme was hy- (v/v)zymepreparationwith cold acetone(20.6 g(-20°C),as protein)and wasthe broughtresulting to pre-30% drolyzed in 6n HC1 containing 2% thioglycolic acid at cipitate was removedby centrifugation. Cold acetone was 105°C for 20~70'hr in an evacuated sealed tube. Before added to the supernatant solution to 70%. The precipitate hydrolysis, norleucine was added to the enzymesolution collected by centrifugation was dissolved in 100ml of to the concentration of 0.5 /miol/mg protein as an internal 10mMpotassium phosphate buffer (pH 7.0) and dialyzed standard. The hydrolyzate was analyzed with an amino for 18hr against 10 liters of the same buffer. acid autoanalyzer (Kyowa Seimitsu Co., type K-101). (ii) DEAE-Sephacel column chromatography. The dia- Cysteine and cystine were determined as cysteic acid after lyzed solution (5.7g as protein) was applied to a DEAE- performate oxidation of the sample according to Hirs.3) Sephacel column (2.5 by 45 cm). The column was washed Tryptophan was determined spectrophotometrically.4) once with 2.5 liters of the equilibration buffer, lOmM The NH2-terminal amino acid was determined by the potassium phosphate (pH 7.0), and then with 2 liters of the dansyl method according to Ikenaka.5) To identify the buffer containing 100mMNaCl. The enzyme was eluted dansyl amino acids produced during acid hydrolysis, thin with a linear gradient of increasing NaCl concentration layer chromatography on Polyamide Layer Sheets between 100 and 300mM (total volume, 2.5 liters). The (Seikagaku Kogyo) was performed. A spotted sheet was active fractions collected and pooled were dialyzed for sequentially developed with four kinds of solvents accord- 24hr against 3 changes of a 10 liter volume of 10mM ing to Woods and Wang.6) The COOH-terminal amino phosphate buffer, pH 7.0, and concentrated to a one- acid was determined with carboxypeptidases (CPase) A tenth volume by ultra filtration with a Minimodule and B (Sigma Chemical Co., type, I-DFP), the molar (Asahikasei Co., Ltd.). ratio of which to the formaldehyde dismutase was 1/ Through the purification procedures, the enzyme was 25.7) The released amino acids were determined with the purified about 22-fold from the cell-free extract of P. autoanalyzer mentioned above. putida F61 (Table I). The enzyme yield after DEAE- Sephacel chromatography was two times higher than that Electrophoresis. Polyacrylamide gel electrophoresis with the previous methods.1* was carried out in 5%polyacrylamide gels in Tris-glycine (Hi) Phenyl-Sepharose CL-4B column chromatography. buffer (pH 8.3) according to Davis.8) The zone of the To the concentrated solution (50ml, 256mg as protein) formaldehyde dismutase activity was visualized by in- was added solid NaCl to a concentration of 4m, and the cubating the gels for 60min at 30°C in 5ml of a reaction mixture was applied to a Phenyl-Sepharose column (2.5 by mixture containing 50mMpotassium phosphate buffer 20 cm) equilibrated with lO mMpotassium phosphate (pH (pH 7.5), 20mM formaldehyde, 0.2mM NAD+, 0.02mg of 7.0) containing 4mNaCl. Elution was carried out with a phenazine methosulfate per ml, 1 mg 2,3,5-triphenyltetra- gradient of decreasing NaCl concentration and increasing zolium chloride per ml, and 2 units of formate dehy- ethylene glycol concentration (the final concentrations drogenase per ml. The reaction was stopped by adding were 0 and 50%, respectively; total volume, 500ml). The 0.5ml of acetic acid and then the gels were removed and elution pattern of the enzyme is described below. The stored in 7% (v/v) acetic acid. The electrophoresis on polyacrylamide gels containing sodium dodecylsulfate withactivea fractionsMinimodule,collectedand dialyzedand pooledforwere24hrconcentratedagainst 3 (SDS) was carried out according to Shapiro et al.9) changes of a 10 liter volume of 10mMpotassium phos- phate buffer (pH 7.0). Gas chromatography. Alcohols, aldehydes and acids were determined by gas chromatography (Shimadzu Preparative polyaerylamide gel electrophoresis. A 0.5-ml GC-8A) with a flame ionization detector. A glass col- aliquot of the enzyme solution (5 mgprotein) obtained at umn (0.26x200cm) was filled with 80/100 mesh Gasu- the Phenyl-Sepharose step was applied to a slab gel (16 by kuropack 54 (Gasukuro Kogyo Inc.). The temperatures 14cm, 2mmthick) which was in contact with a cooling of the column and inlet were maintained at 180 and plate, and electrophoresis was carried out at 15mAac- 200°C, respectively. The flow rate of nitrogen carrier cording to Bringer et al.10) After the electrophoresis, gas was 60ml/min. The reaction mixture was directly in- the gel was blotted with a filter paper (Toyo Roshi No. jected into the column. 51A), which was immediately stained with Coomassie brilliant blue. The gel zone corresponding to the stained Enzyme purification. As described previously,1* the en- protein band on the paper was cut out and crushed with zyme waspurified from the cell-free extract of P. putida a teflon homogenizer. From the disintegrated gel, the en- F61 by protamine treatment, ammoniumsulfate fraction- zyme was eluted with 10mMphosphate buffer (pH 7.0) ation, and chromatographies on Phenyl-Sepharose CL- with stirring overnight. The polyacrylamide was remov- 4B, DEAE-Sephacel and hydroxyapatite. In order to ed by centrifugation, and the supernatant wasdialyzed Formaldehyde Dismutation Catalyzing Enzyme 2019

Table I. Purification of the Formaldehyde Dismutase from Pseudomonas putida F61 Total Total Specific Step protein activity activity Purification f (mg) (U) (U/mg) ( /o) Cell-free extract 21 ,400 90,000 4.21 1.0 100 Protamine sulfate 20,600 87,200 4.23 1.0 96.9 Acetone fractionation 5,700 52,600 9.23 2.2 58.4 DEAE-Sephacel 256 24, 100 91. 1 21.6 26.8 against lOmMphosphate buffer (pH 7.0), and then con- centrated with an Immersible CX-10 ultra filter (Millipore Corp.) to 0.5ml.

RESULTS Protein nature of the purified enzyme As shownin Fig. 1, two active fractions of formaldehyde dismutase appeared on Phenyl- Sepharose column chromatography as de- ; v...^ 100 150 scribed under Materials and Methods. The 20 * å : 10ml/fraction first fraction (A; fractions 104~130) was Fig. 1. Phenyl-Sepharose CL-4B Column Chromatog- identical with the enzyme which was purified raphy of the Formaldehyde Dismutase. previously. The specific activity of the latter The details are given in Materials and Methods. ----, fraction (B; fractions 183-234), 92/miol/ NaCl; - --, ethylene glycol. minà"mg,was about half of that of the A-frac- tion. On polyacrylamide gel electrophoresis, the A-fraction preparation gave two distinct Molecular weight and subunit of the B-fraction protein bands (A-l and A-2) whose migra- enzyme tion rates were 0.38 and 0.44, respectively. The molecular weight of the B-fraction en- Onthe other hand, the B-fraction gave a sin- zyme was estimated to be 2.2xlO5 by gel gle band whose migration rate was identical filtration on Bio-Gel A-1.5m and the molec- with that of A-2. Since each protein band ular weight of its subunit to be 5.5x 104 by coincided with enzyme activity, all the three SDS gel electrophoresis. These values are fractions (A-l, A-2 and B) were considered to consistent with those of the A-fraction de- be active formaldehyde dismutase. It was ob- scribed previously.X ) served that two activity bands were detected also on disc gel electrophoresis of the cell- COOH-terminal amino acid of the enzyme free extract of the bacterium. Each active en- After 2hr-hydrolysis with CPase A, 0.24 zyme fraction was prepared by preparative mol of lysine per mol of the substrate en- polyacrylamide gel electrophoresis as describ- zyme (B-fraction) was found to be released. ed under Materials and Methods. These Since COOH-terminal lysine was reported to be three fractions gave single bands on SDS- released slowly by CPase A,n) the hydrolysis polyacrylamide gel electrophoresis, and their was carried out with a mixture of CPase A and migration rates (0.52) were identical. Further- B (1:1 w/w) for 2hr. In this hydrolyzate, more, the NH2-terminal amino acid of each 0.92mol of lysine per mol of the enzymewas enzyme fraction was found to be arginine. found. Small amounts of glycine and serine These facts indicate that the three enzyme (0.21 and 0.04mol/mol of enzyme) were also species are an identical polypeptide. detected in the hydrolyzate. From these re- 2020 N. Kato et al.

Table II. AminoAcid Composition of the characteristic absorbance at 320 nm.1* The ab- Formaldehyde Dismutase sorption spectrum was the same before and Details are given in Materials and Methods. after treatment with the reducing reagents. It is at present unclear whether the modification . . ., Residues/ A . ., Residues/ Ammoacid u ' Ammoacid , . caused by such inhibitors occurred at an subumt subunit amino acid residue(s) of the enzymeprotein or Asp 72 He 30 Thr 1 5 Leu 45 at the unknownprosthetic group. Mercury(II) Ser 9 Tyr 1 5 chloride noncompetitively inhibited the en- Glu 40 Phe 1 4 zyme and its Ki was 0.2 /im. The reversibility of Pro 28 Lys 30 this inhibition was examined by adding 2.5 mM Gly 64 NH3 73 Ala 45 His 1 5 dithiothreitol and 2.5 /im 2-mercaptoethanol, Cys 9 Arg 22 respectively, to the enzyme after treatment Val 47 Trp 4 with 0.01 mM HgCl2 (94% inhibition). After Met 7 incubation for lOmin at 30 °C, almost the full enzyme activity was seen. This indicates that the enzyme contains a reactive sulfhydryl suits, the sequence of the COOH-terminal group. end of the enzyme was assumed to be -Ser- Gly-Lys. Cross-dismutation reaction The substrate specificity of the enzyme in the Aminoacid composition of the enzyme cross-dismutation reaction was examined. The The amino acid composition of the enzyme equation for the overall reaction of cross- (B-fraction) based on a minimal polypeptide dismutation is as follows; chain of a molecular weight of 5.5x104 is RCHO+H2O+R CHO > given in Table II. The low content of tryp- RCOOH+R CH2OH tophan seems to be a characteristic of the In this experiment, formaldehyde was used as enzyme. This reflects presumably the low ex- one substrate (R=H) and the other (R/CHO) tinction coefficient of the enzyme (^lo nm)- was varied. The enzyme has been found to No carbohydrate was detected in the pu- strongly catalyze the dismutation of formal- rified enzyme preparations by the orcinol- dehyde alone. In order for the cross-dis- H2SO4 and phenol-H2SO4 reactions.12) mutation to proceed preferentially, the con- centration of R/CHO in the reaction mix- Enzymeinhibition The enzyme was preincubated with indi- ture was made a two-fold excess of that vidual inhibitors at several concentrations at theoretically required. As shown in Table III, the enzyme was active toward propional- 30°C for 30min, and then the remaining ac- dehyde, acrolein, butyraldehyde, isobutyral- tivity was determined under the standard assay conditions. The inhibitor constant (Ki) was dehyde and crotonaldehyde. Under these con- ditions, aldehyde (R/CHO) was exclusively determined by the graphical method of reduced to the corresponding alcohol and an Dixon.13) The enzyme (B-fraction) was inhibit- equimolar amount of formaldehyde was oxi- ed by carbonyl reagents; 1 mMsemicarbazide (67.3% inhibition) and l mMhydroxylamine dized to formic acid. When higher aldehydes, (81.8%). Semicarbazide was found to be a such as methacrolein, valeraldehyde and hex- noncompetitive inhibitor and its Ki was aldehyde, were used as one substrate (R/CHO), and formic acid were 0.52 mM.The enzyme was susceptible to reduc- detected as products. Since these aldehydes ing reagents; 0. 1 % sodium borohydride (37.2% inhibition) and sodium thiosulfate (32.4%). are scarcely soluble in aqueous solution, the The enzyme has been reported to show a concentration of aldehydes dissolved in the Formaldehyde Dismutation Catalyzing Enzyme 2021

Table III. Cross-dismutation between A Formaldehyde and Aldehyde by the Formaldehyde Dismutase The reaction mixture contained 50mM potassium phosphate buffer (pH 7.0), 10mMformaldehyde, 20mM RCHOand 0.5 units of the formaldehyde dismutase per ml, and was incubated at 30°C for 30min. Substrate o) (in din (iv) Fig. 2. Gas Chromatographic Analysis of the RCHO RCH2OH Alcohol:Aldehyde Oxidoreduction Reaction by the Formaldehydel liJLJLDismutase. Propionaldehyde Acrolein The reaction mixture contained 50niMpotassium phos- Butyraldehyde phate buffer (pH 7.0), 10mM «-butanol (A), 10mM iso- butyraldehyde (B) and 8units of the formaldehyde dis- Isobutyraldehyde Crotonaldehyde mutase per ml, and was incubated at 30°C. (I); 0 time and Methacrolein minus enzyme, (II); after 60 min incubation, (III); minus Valeraldehyde isobutyraldehyde, (IV); minus «-butanol.

PropanolAllylButanolIsobutylCrotylalcoholHexaldehyde

60 - reaction mixture was muchlower than that of formaldehyde. Therefore, dismutation of formaldehyde itself proceeded predominantly. 5i / !Ȉ"/ Alcohol : aldehyde oxidoreduction The enzymewas found to catalyze a coupled oxidoreduction between an alcohol and an al- oLd 1 1 1 1-_i l_ dehyde without addition of an electron accep- 0 60 120 tor. In this reaction, alcohol oxidation to the Reaction period (min) corresponding aldehyde was accompanied by Fig. 3. Time Course of the Alcohol: Aldehyde Oxido- aldehyde reduction to alcohol. The alcohol reduction Reaction. The reaction conditions were the same as described in Fig. and aldehyde are exchanged for each other in 2. the one oxidoreduction reaction; RCH2OH+R CHO- >RCHO+R CH2OH amounts of butyraldehyde and isobutyl al- Figure 2 shows an example of the alcohol: cohol formed were the same as those of the aldehyde oxidoreduction reaction catalyzed corresponding alcohol and aldehyde consum- by the enzyme. After 60min reaction, butyr- ed, respectively. The substrate specificity of aldehyde (P) and isobutyl alcohol (Q) were the oxidoreduction is shown in Table IV. Al- observed to be formed from n-butanol (A) and though the conversion ratio changed with the isobutyraldehyde (B). No or little change of combination of alcohol and aldehyde, ethanol, the substrate was observed in the reaction propanol, allyl alcohol, w-butanol and iso- mixtures containing one substrate only, and butyl alcohol, the corresponding aldehydes the reaction did not occur without addition of were reactive in every combination. Alipha- the enzyme. As shown in Fig. 3, the conversion tic alcohols with longer carbon chains than ratio of the alcohol : aldehyde oxidoreduction C-5, secondary alcohols, diols and aromatic (fl-butanol + isobutyraldehyde->butyralde- alcohols and the corresponding aldehydes hyde-fisobutyl alcohol) reached 55%. This were not reactive in this reaction. Methanol reaction proceeded stoichiometrically, i.e., was not utilized in the reaction. It is thought during the course of the reaction, the molar that the redox potential for the methanol/ 2022 N. Kato et al.

Table IV. Alcohol : Aldehyde Oxidoreduction Reaction by the Formaldehyde Dismutase The reaction mixture contained 50 mMpotassium phosphate buffer (pH 7.0), 10 mMeach of RCH2OHand R'CHOand 8 units of the formaldehyde dismutase per ml. Figures denote conversion ratios after 60 min reaction at 30°C. RCHO RCH2OH - Acetaldehyde Propionaldehyde Acrolein Butyraldehyde Isobutyraldehyde

Methanol 0 0 0 0 0 Ethanol * 51.3 32.6 25.8 54.5 Propanol 41.0 * 49.6 50.4 50.4 Allyl alcohol 64.6 52.3 * 53. 1 68.7 Butanol 41.7 44.7 38.8 * 48.0 Isobutyl alcohol 27.7 33.6 17.6 55.4 *

formaldehyde half reaction is insufficient to OH reduce other aldehydes.14) \>H ^O (1) DISCUSSION In this work, the enzyme was found to cata- Two forms of formaldehyde dismutase dis- lyze a novel alcohol : aldehyde oxidoreduction tinguishable on disc gel electrophoresis were reaction: found in a purified enzyme preparation. If the H appearance of two active bands on electro- R-cr phoresis is normal behavior of the natural +*o enzyme (although this may be an artifact), the B-fraction enzyme is thought to undergo a H modification during the hydrophobic interac- s + R-C OH à"^ O tion chromatography. The irreversible modifi- \ (2) cation maybe a conformational change of the Reaction (2) is analogous to the cross-dis- enzymeprotein, and does not affect the cata- mutation, i.e., the hydrated aldehyde (in Eq. lytic activity definitely. Catalytic properties of 1) and alcohol (in Eq. 2) are oxidized to an the B-fraction were the same as those of acid and aldehyde, respectively, coupling with fractions A-l and A-2, and a mixture of them, the reduction of non-hydrated aldehyde. although the specific activity of the B-fraction From the above consideration, this enzyme was half that of the A-fraction. should be classified as an alcohol dehydro- Preferable substrates for the dismutation genase or alcohol : aldehyde were aldehydes that are hydrated to a great (EC 1.1.99.X). extent, such as formaldehyde, acetaldehyde and methylglyoxal.1* Thus, the dismutation Acknowledgments. This work was supported in part reaction can be seen as a coupled oxidoreduc- by a Grant-in-Aid for Scientific Research from the tion between an aldehyde and an "alcohol" Ministry of Education, Science and Culture of Japan. formed by the hydration. The existence of a REFERENCES hydrated form of aldehyde was also indispens- able for the cross-dismutation: 1) N. Kato, K. Shirakawa, H. Kobayashi and C. Sakazawa, Agric. Biol. Chem., 47, 39 (1983). 2) R. H. Abeles and H. A. Lee, Jr., /. Biol. Chem., 235, 1499 (1960). ^O H^ OH 3) C. H. Hirs, J. Biol. Chem., 219, 611 (1956). Formaldehyde Dismutation Catalyzing Enzyme 2023

H. Faenkel-Conrat, "Methods in Enzymology," Vol. Biophys. Res. Commun., 28, 815 (1967). IV, ed. by S. P. Colowick and N. O. Kaplan, S. Bringer, B. Sprey and H. Sahm, Eur. J. Biochem., Academic Press Inc., New York, 1957, p. 247. 101, 563 (1979). T. Ikenaka, "Seikagaku Jikken Koza," Vol. l-II, ed. R. P. Amber, "Methods in Enzymology," Vol. XI, by K. Narita and T. Murachi, Tokyo Kagaku Dojin, ed. by C.H.W. Hirs, Academic Press, New York, Tokyo, 1976, p. 142. 1967, p. 155. K. P. Woods and K. T. Wang, Biochim. Biophys. R. J. Winzler, "Methods of Biochemical Analysis," Ada, 133, 369 (1967). Vol. II, ed. by G. Glick, Interscience, New York, T. Ikenaka, "Seikagaku Jikken Koza," Vol. l-II, ed. 1955, p. 279. by K. Narita and T. Murachi, Tokyo Kagaku Dojin, Tokyo, 1976, p. 203. M. Dixon, Biochem. /., 55, 170 (1953). C. Anthony, "The Biochemistry of Methylotrophs," B. J. Davis, Ann. N. Y. Acad. ScL, 121, 404 (1964). Academic Press, London, 1982, p. 154. A. L. Shapiro, E. Vinuela and J. V. Maizel, Biochem.